Abstract Immunotherapy is one of the most promising new cancer treatment approaches. While some patients experience astounding results in long-term remission rates and eradication of metastatic growths, the number of responders is surprisingly low. Due to the large number of coincident factors and cell types, the predominant way that researchers study tumors and their responses to immunotherapies is through ex vivo analyses, such as histology or flow cytometry. Unfortunately, these techniques preclude longitudinal studies because the tissue must be excised, making it very challenging to study the dynamic nature of the tumor over time and resulting in a slow and costly research pipeline. While noninvasive imaging technologies exist, multiplexing several tumor readouts is either not possible or requires a highly complex workflow. To address this need, SonoVol is proposing to build and validate a novel preclinical molecular imaging platform, capable of multiplexing several parameters of interest (multiple T cell subtypes, tumor cells, etc.) with 3D tomographic imaging. The system will leverage ultrasound/photoacoustic (USPA) imaging in combination with a novel genetic reporter - bacterial phytochrome photoreceptors (BphP). BphPs are a class of proteins that exhibit near-infrared (NIR) absorbance and fluorescence and are red-shifted relative to any other phytochromes, making them ideal for in vivo imaging at depth. Additionally, BphPs experience pronounced conformational changes upon photoisomerization, enabling photo-control and, consequently, highly sensitive cell tracking (tens to hundreds of cells at depth). By commercializing this imaging/reporter biotechnology within the SonoVol VegaTM, we will enable researchers to perform mechanistic studies of tumor microenvironment and accelerate translation of critical immunotherapies to the clinic. This work will proceed in three stages. First, we will deploy new software modules that enable pushbutton PA-BphP imaging on the Vega. Second, we will prepare and verify two distinct BphP-labelled cells (murine prostate tumor cells and CD8+ T cells). Finally, we will perform a validation of our imaging system using adoptive transfer and observe T cell infiltration into tumors under two immunotherapy conditions; anti-PDL1 therapy alone, and a combination therapy shown to enhance T cell trafficking (anti-PDL1 plus radiation).